Was performed as described68 (link). For Flag detection, DYKDDDDK Tag Rabbit antibody was used at 1:100 dilution (Cell Signaling Technology, 14793). Where indicated, 30 min before fixation with 4% paraformaldehyde, cells were incubated with 100 nM MitoTracker Red CMXRos (Invitrogen, M7512) to assess mitochondrial membrane potential. Mounted coverslips were imaged on a Leica TCS SP8 Confocal Laser Scanning Microscope (Leica Microsystems) with ×63 objective and 1.4 numerical aperture. Quantification of MitoTracker Red CMXRos fluorescence intensity per cell was performed using ImageJ (NIH) and expressed as mean integrated density. For detection of BODIPY FL C16 in vivo, sections from freshly isolated livers were mounted with Fluoromount-G medium (SouthernBiotech, 0100) and imaged on Leica TCS SP8 Confocal Laser Scanning Microscope with ×10 objective and 1.4 numerical aperture.
Tcs sp8 confocal laser scanning microscope
Manufactured by Leica
Sourced in Germany, United States, Japan, United Kingdom, China
The Leica TCS SP8 is a confocal laser scanning microscope. It is designed to provide high-resolution imaging of biological samples. The system utilizes laser excitation and confocal detection to capture detailed images with improved contrast and optical sectioning capabilities.
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Lab products found in correlation
Las x software, by Leica (20 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (9 mentions)
Las x, by Leica (9 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Application suite x las x software, by Leica (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Las af lite software, by Leica (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Las af software, by Leica (3 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Goat serum albumin, by Beyotime (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (3 mentions)
452 protocols using tcs sp8 confocal laser scanning microscope
1
Quantifying Mitochondrial Membrane Potential
2
Immunofluorescent Staining of Lung Tissue and Macrophages
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Frozen sections of lung tissue were placed at room temperature for 30 min, lung cells were fixed with 4% PFA for 15 min, permeabilized with 0.2% Triton X‐100 for 10 min, then blocked with 3% BSA for 1 h and washed 3 times in PBS. The lung tissues were then incubated with rabbit anti‐STING (1/300) overnight at 4°C, washed and incubated with Goat anti‐Rabbit IgG (H+L) Alexa Fluor 568 secondary antibody for 1 h at room temperature. After washing 3 times, lungs were incubated with DNA dye Draq5 (1/1000) for 5 min, washed and quenched before mounting. Finally, tissues were observed using Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems Ltd., Wetzlar, Germany). Images were analyzed using LAS X software.
In vitro, BMDMs were harvested and placed onto cell slides overnight at 37°C 5% CO2 in DMEM complemented medium. Cells were fixed with 4% PFA for 20 min, permeabilized with 0.2% Triton X‐100 for 5 min, blocked with 3% BSA for 1 h, washed with PBS, and incubated with MitoTracker Red (500 nM) for 45 min at 37 °C. After washing, cells were stained with DNA dye Draq5 (1/1000) for 5 min to label dsDNA and the anti‐fluorescence quencher was added before mounting. Cells were then visualized via Leica TCS SP8 laser scanning confocal microscope.
In vitro, BMDMs were harvested and placed onto cell slides overnight at 37°C 5% CO2 in DMEM complemented medium. Cells were fixed with 4% PFA for 20 min, permeabilized with 0.2% Triton X‐100 for 5 min, blocked with 3% BSA for 1 h, washed with PBS, and incubated with MitoTracker Red (500 nM) for 45 min at 37 °C. After washing, cells were stained with DNA dye Draq5 (1/1000) for 5 min to label dsDNA and the anti‐fluorescence quencher was added before mounting. Cells were then visualized via Leica TCS SP8 laser scanning confocal microscope.
3
Cellular Immunofluorescence Staining of Macrophages
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In cellular immunofluorescence staining, after 30 min of incubation of Dil-ADNVs with MH-S cells, Chlorpromazine hydrochloride (CPZ) at 10 µg/mL or Genistein at 200 µM (all from Aladdin, Shanghai, China) was added. After 4 h, samples were collected, and cells were fixed with 4% paraformaldehyde and washed thrice with PBS. The sections were blocked with 3% BSA for 1 h, washed 3 times with PBS, and treated with YF® 488-phalloidin overnight at 4 °C, washed 3 times with PBS, and incubated with Alexa Fluor 488 goat antibody for 1 h at room temperature, washed 3 times with PBS, and DAPI (1: 1000, Beyotime, Shanghai, China) were stained for 10 min, washed with PBS, and the cells were observed using a Leica TCS SP8 laser scanning confocal microscope after addition of an anti-fluorescence quenching agent. Primary rabbit antibody against F4/80 (1:200, Abcam, USA) was incubated overnight with frozen lung tissue sections at 4 °C, and Alexa Fluor 488 goat antibody was incubated for 1 h at room temperature. Nuclei were stained with DAPI (1:1000, Beyotime, Shanghai, China), washed three times with PBS, and imaged using a Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).
4
Quantifying Microglial Cells in Brain Tissue
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Frozen brain tissues were blocked with 10% goat serum in 0.01 M PBS for 1 h and then incubated overnight at 4 °C with primary antibodies (Abs). Brain sections (20 μm) were incubated with the following primary Abs: mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel) and rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan). The brain sections were then washed with 0.01 M PBS and incubated with the respective Alexa Fluor® 488- or 568-conjugated secondary Abs (1:500; Invitrogen Corporation). Fluorescent images were acquired using a TCS SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). For imaging acquisition, a prescan of all samples was performed to ensure confocal settings below saturation. For each experiment, all images were obtained using the same confocal settings. Six slices at 120 μm intervals from each brain were used to examine Iba1-positive cells. Three microscopic fields (0.01 mm2) were randomly selected in each slice with the same reference position for quantification. The Iba1-positive cell number was counted in a blinded manner, and the area was measured by Leica LAS AF Lite software (Leica, Germany).
5
Visualizing Lignin Changes in Pear Infected by Botryosphaeria
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The Conference pear plantlets were inoculated with B. dothidea spore suspension or double‐distilled water (mock‐inoculated control). Stem segments infected with B. dothidea just under the edge of lesions at different timepoints and mock‐inoculated controls were fixed in formalin‐acetic acid‐alcohol for 2 days, dehydrated with an ethanol gradient, and embedded in melted wax, then 4‐μm cross‐sections were cut using an RM2016 ultramicrotome (Leica Microsystems). Cross‐sections were stained with toluidine blue O (0.5% wt/vol) then observed using an Eclipse 80i microscope (Nikon). Three plantlets infected with B. dothidea and mock‐inoculated controls at each timepoint were collected and processed. The width of xylem in mock and B. dothidea‐infected plantlets was measured by ImageJ software according to the toluidine blue O‐stained sections. The xylem width was the average value of 18 sections from three plantlets at each timepoint.
To further visualize autofluorescence from total lignin, sections with a diameter of 4 μm were observed using a TCS‐SP8 confocal laser scanning microscope (Leica Microsystems) excited with a 405 nm laser, and emission signals were captured at 460 nm (Vanholme et al., 2013 (link)).
To further visualize autofluorescence from total lignin, sections with a diameter of 4 μm were observed using a TCS‐SP8 confocal laser scanning microscope (Leica Microsystems) excited with a 405 nm laser, and emission signals were captured at 460 nm (Vanholme et al., 2013 (link)).
6
Immunofluorescence Microscopy for Cellular Analysis
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For immunofluorescence microscopy, cells were rinsed with PBS and fixed with 4% paraformaldehyde and then blocked with 1% horse serum (Gibco, 16050122) in PBS. Samples were incubated with appropriate primary antibodies at 4 °C overnight and followed by incubation with secondary Alexa-Flour 488, 596 dye-labeled antibodies. The nuclei were stained with 1 μg/ml DAPI in PBS for 10 min. For BODIPY staining, cells were loaded with 10 μg/ml BODIPY 493/503 in PBS for 20 min. Coverslips were mounted using ProLong Gold Antifade Reagent (Life Technologies, P36930) and images were captured with TCS SP8 confocal laser scanning microscope (Leica Microsystems). The confocal images were acquired using identical exposure settings on the same day in each experiment replicate. 5 images were randomly selected and quantified using Image J software.
7
Evaluating Cell Growth and Morphology
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The growth of WT and mutant strains used in this study was evaluated under the conditions previously described16 (link). Briefly, cells were grown under 30 or 100 μmol photons m−2 s−1 of continuous white fluorescent light in HS media, and cells were counted every 24 h. To assess cell shapes, cells at the steady-state growth phase (day 6) were imaged at room temperature using a TCS SP8 confocal laser scanning microscope (Leica Microsystems, Germany) equipped with a HC PL APO CS2 63×/NA1.40 oil objective lens. A 448-nm diode laser with 2% output was selected to obtain bright-field images. The images were produced on a PMT-based detector. All images were acquired at a 600-Hz laser scan speed and analyzed by LASX software (Leica Microsystems).
8
Root Anatomy of Rs-Infected Plants
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Roots of noninfected and Rs-infected plants of all cultivars were collected at 6 dpi (n=5 per treatment and cultivar). A 10-mm long section was excised from all the roots at the middle region (approximately 5 cm from the root tip). Root sections were fixed in 60 mM phosphate buffer (pH 7.2) containing 4 w% formaldehyde, washed, dehydrated in a series of ethanol solutions, and gradually infiltrated with LR White resin (Agar Scientific Ltd., Stansted, UK). The resin was polymerized at 55°C for 48 h. Semi-thin (1-µm) cross sections were cut from the resin blocks, using an Ultracut-E microtome (Reichert-Jung, Heidelberg, Germany) and stained with safranin O (Searle Diagnostic, High Wycombe, UK) for cell wall, with Fast Green FCF (Sigma-Aldrich) applied as counterstain. The stained sections were mounted in 50 v% glycerol and examined with a TCS SP8 confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany). Safranin O was excited at 514 nm, and signals were detected at 600 to 720 nm. Fast Green FCF was excited at 633 nm, and signals were detected at 640 to 780 nm. Micrographs were taken using the Leica Advanced Fluorescence software v3.1.5.1638 (Leica Microsystems) with no further processing of the images.
9
Generating pTCH4:TCH4-GFP Transgenic Plants
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In order to construct the pTCH4:TCH4-GFP plasmid, the promoter and genome of TCH4 were amplified via PCR with the TCH4-GFP-1 and TCH4-GFP-2 primers successively (Supplementary Table S1 ) and then inserted into the rebuilt PBI121 vector at ScaI and SmaI restriction sites. pTCH4:TCH4-GFP transgenic plants were generated via the Agrobacterium-mediated floral-dip method [46 (link)]. For GFP observation, plants soaked in 4 μM FM4-64 with or without 0.4 g/mL sucrose for plasmolysis were observed by a Leica TCS SP8 confocal laser scanning microscope (www.leica-microsystems.com/home/ accessed on 1 November 2021). For GFP observation in Nicotiana benthamiana leaves, the pTCH4:TCH4-GFP and PIP2A-mCherry plasmids [48 (link)] co-injected into tobacco leaves soaked with or without 0.4 g/mL sucrose for plasmolysis were inspected by confocal laser scanning microscopy.
10
Visualizing Mitochondrial Apoptosis Pathways
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For Confocal imaging, U2OS BAX−/−BAK−/− cells seeded on coverslips were transfected with (Halo-BOK, GFP-BOK or GFPBOK∆C) and (GFPSEC61 or mCherry-SEC61) and then treated with 10 µM QVD. 16 h after transfection, the cells were incubated with 150 nM MitoTracker Deep Red (Thermo Fisher) and HaloTag TMR Ligand (Promega) (when transfecting with Halo-BOK) for 20 min at 37 °C. The cells were then washed 3 times with fresh media and were fixed using Paraformaldehyde. Imaging was performed on a TCS SP8 confocal laser scanning microscope (Leica Microsystems) equipped with a PL Apo 63x/1.40 Oil CS2 objective and a tunable white light laser (470–670 nm). The signal was acquired with sensitive HyD detectors (Leica Microsystems).
For STED imaging, U2OS BAX−/−BAK−/− cells seeded on coverslips were transfected with Halo-BOK and Smac-GFP and then treated with 10 µM QVD. 16 h after transfection, the cells were incubated with 0.3 μM Janelia Fluor 549 HaloTag Ligand (Promega) and 150 nM MitoTracker Deep Red (Thermo Fisher) for 20 min at 37 °C. The cells were then washed 3 times with fresh media and were fixed using Paraformaldehyde. Images were acquired using TCS SP8 gSTED microscope (Leica Microsystems) equiped with HL PL APO 100x/1.40 Oil STED, a tunable white light laser (470–670 nm) and 750 nm depletion laser. The signal was acquired with sensitive HyD detectors (Leica Microsystems).
For STED imaging, U2OS BAX−/−BAK−/− cells seeded on coverslips were transfected with Halo-BOK and Smac-GFP and then treated with 10 µM QVD. 16 h after transfection, the cells were incubated with 0.3 μM Janelia Fluor 549 HaloTag Ligand (Promega) and 150 nM MitoTracker Deep Red (Thermo Fisher) for 20 min at 37 °C. The cells were then washed 3 times with fresh media and were fixed using Paraformaldehyde. Images were acquired using TCS SP8 gSTED microscope (Leica Microsystems) equiped with HL PL APO 100x/1.40 Oil STED, a tunable white light laser (470–670 nm) and 750 nm depletion laser. The signal was acquired with sensitive HyD detectors (Leica Microsystems).
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